Pseudomonas putida is a bacterium which can grow on d-camphor as its source of carbon and energy. In the presence of camphor this organism contains a soluble cytochrome P-450 type of monooxygenase system. The membrane-bound electron transport system which occurs in this organism is unusual in that the terminal oxygen reducing enzyme is not a heme "a" type of cytochrome. The ultimate goal of this research program is to understand the mechanism of oxygen reduction by these two systems and their interaction. RESEARCH GOALS FOR THE COMING YEAR: A. Studies of the sequence of electron carriers in the membrane-bound electron transport system of P. putida will be carried out using the stopped flow spectrophotometer which was developed for the study of turbid suspensions. The kinetics of reduction and reoxidation of these hemeproteins in the presence and absence of selected inhibitors should permit us to begin to understand the interrelationships of electron carriers in this unusual electron transport system. These studies will be complemented with appropriate measurements of oxygen uptake and low- and room-temperature spectroscopy. B. The studies of the effect of cations on substrate binding to cytochrome P-450 are almost complete and will be extended to studies of the effect of putidaredoxin on substrate binding and the effect of various salts on this reaction. In addition to optical absorption and EPR-spectroscopy we will examine these protein-protein interactions using gel exclusion chromatography. C. The studies of carbon monoxide binding by infrared spectroscopy have been very fruitful in understanding the reaction of this ligand with other hemeproteins. We will extend these results reported here using a variety of salt, pH and substrate conditions. We will also attempt to look directly at the interaction of oxygen with the heme iron of cytochrome P-450 using infrared spectroscopy. D. As usual, wherever appropriate, we will extend the results obtained with the bacterial cytochrome P-450 to the mammalian drug oxidation reactions. BIBLIOGRAPHIC REFERENCE: Griffin, B.W., Peterson, J.A., Estabrook, R.W. (1977) in "Porphyrins" (Dolphin, D., ed.) "Cytochrome P-450: Biophysical Properties and Catalytic Function", in press.